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Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system

ABSTRACT: Summary Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020, 2021). Graphical abstract Highlights • Measure nuclear mRNA export by fluorescent in situ hybridization (FISH)• Examine protein import and export activities with a dual reporter system• Use an open-source software to quantify images• Examine and quantify NCT activities in cultured cells, including neurons Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells.

SUBMITTER: Cui H

PROVIDER: S-EPMC9641092 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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